Exploitation of lignocellulosic biomasses for production of biofuels, biochemicals, and pharmaceuticals is alternative to the world's limited fossil resources. Bio refineries should replace oil refineries, producing the same products, but using renewable resources: lignocellulosic biomasses. Lignocellulosic biomasses mainly consist of cellulose, hemicelluloses, and lignin, with different distribution of each component depending on the specific plant species, from which it is derived. Cellulose is of great interest in terms of creating a sugar platform for biofuels and chemicals as its hydrolysis product, glucose, can readily be fermented into ethanol or converted into high value chemicals. Cellulose is a polymer of the simple sugar glucose covalently bonded by beta-1,4-glycosidic linkages. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. The complete hydrolysis of cellulose involves the synergistic action of cellobiohydrolases (EC 3.2.1.91), endoglucanases (EC 3.2.1.4), and beta-glucosidases (EC 3.2.1.21). The cellobiohydrolases are capable of degrading the crystalline parts of cellulose by cleaving off cellobiose molecules from the ends of the cellulose chains. The endoglucanases digest the cellulose polymer at random locations, hydrolyzing glucosidic bonds of the more amorphous regions of the cellulose, decreasing the degree of polymerization and opening it to attack by cellobiohydrolases by creating more free ends for attack by cellobiohydrolases. Finally, the beta-glucosidases act in the liquid phase hydrolyzing mainly cellobiose (a water-soluble beta-1,4-linked dimer of glucose) to glucose, but also to some extent cellodextrins, sugars with a low degree of polymerization.
Historically, enzymes from Trichoderma reesei and Aspergillus niger are known as a good match for the hydrolysis of cellulose; T. reesei enzymes mainly contributing with cellobiohydrolase and endoglucanase activity and A. niger enzymes with beta-glucosidase activity (Sternberg D et al. Can J Microbiol 1977; 2:139-47). Beta-glucosidases are of key importance as they are needed to supplement the cellobiohydrolase and endoglucanase activities for final glucose release and at the same time decreasing the accumulation of cellobiose and shorter cellooligmers that are known as product inhibitors for the cellobiohydrolases (Zhang Y-P et al. Biotechnol Adv 2006; 5:452-81). Especially efficient beta-glucosidases, that are not themselves easily inhibited by their substrate, glucose, are of great interest. Currently, most commercial cellulase preparations are produced by T. reesei, e.g. Celluclast 1.5 L (Novozymes A/S), which has to be supplemented with extra beta-glucosidase activity from another source, e.g. Novozym 188 (Novozymes A/S), in order to improve cellulose hydrolysis. However, the commercial available beta-glucosidases have relatively low long-term temperature stability. Robustness, thermostability and substrate specificity are very important characteristics for enzymes to be applied in industrial processes.
The conversion of cellulosic feedstocks into bioethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily processed, for example fermented by yeast into ethanol. Since glucose is readily fermented to ethanol by a variety of yeasts while cellobiose is not, any cellobiose remaining at the end of the hydrolysis represents a loss of yield of ethanol. More importantly, cellobiose is a potent inhibitor of endoglucanases and cellobiohydrolases. The accumulation of cellobiose during hydrolysis is extremely undesirable for ethanol production. Other than biofuels, the monomeric sugars (including glucose) produced by enzymatic hydrolysis in the biorefinery will be used as a platform for biochemicals, plastics, pharmaceuticals, etc.
Cellobiose accumulation has been a major problem in enzymatic hydrolysis because cellulase-producing microorganisms produce little beta-glucosidase. The low amount of beta-glucosidase results in a shortage of capacity to hydrolyze the cellobiose to glucose. Several approaches have been used to increase the amount of beta-glucosidase in cellulose conversion to glucose.
Thus it would be an advantage in the art to provide beta-glucosidases with improved properties for converting cellulosic materials to polysaccharides, cellodextrins, disaccharides and monosaccharides. Improved properties include altered temperature-dependent activity profiles, thermostability, pH activity, pH stability, and substrate specificity.